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Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation

Henrik ten Freyhaus , Michael Huntgeburth , Kirstin Wingler , Jessika Schnitker , Anselm T. Bäumer , Marius Vantler , Mohamed M. Bekhite , Maria Wartenberg , Heinrich Sauer , Stephan Rosenkranz
DOI: http://dx.doi.org/10.1016/j.cardiores.2006.01.022 331-341 First published online: 15 July 2006

Abstract

Objective Reactive oxygen species (ROS) produced by NAD(P)H oxidases (Nox) play a significant role in the pathophysiology of cardiovascular diseases. Expression and activity of NAD(P)H oxidases are regulated by growth factors such as angiotensin II and platelet-derived growth factor (PDGF). We characterized the effects of the novel Nox inhibitor VAS2870 on PDGF-dependent ROS liberation and cellular events in vascular smooth muscle cells (VSMC).

Methods and results PDGF-BB increased NAD(P)H oxidase activity (lucigenin-enhanced chemiluminescence) and intracellular ROS levels (detected by confocal laserscanning microscopy using 2,7-DCF) to 229±9% and 362±54% at 1 and 2 h, respectively. Preincubation with VAS2870 (10 and 20 μM) completely abolished PDGF-mediated NAD(P)H oxidase activation and ROS production. Since ROS are involved in various growth factor-induced cellular functions, the influence of VAS2870 on PDGF-induced DNA synthesis and chemotaxis was determined. PDGF promoted a 4.2±0.2-fold increase of VSMC migration (modified Boyden chamber, p<0.01) and increased DNA synthesis by maximally 3.2±0.4-fold (BrdU incorporation, p<0.01) in a concentration-dependent manner. Preincubation with VAS2870 (0.1–20 μM) did not affect PDGF-induced cell cycle progression. However, it abolished PDGF-dependent chemotaxis of VSMC in a concentration-dependent manner (100% inhibition at 10 μM). These findings were related to PDGF-dependent signaling events. Western blot analyses using phospho-specific antibodies revealed that the downstream signaling molecules Akt, Erk, and Src were activated by PDGF. However, VAS2870 blocked PDGF-dependent activation of Src, but not of Akt and Erk, in a concentration-dependent manner.

Conclusions VAS2870 effectively suppresses growth factor-mediated ROS liberation in VSMC. Furthermore, it completely inhibits PDGF-dependent VSMC migration, whereas it does not affect DNA synthesis. These divergent effects reflect the critical role of Src activity, which–in contrast to Akt and Erk–appears to be redox-sensitive and is absolutely required for PDGF-induced chemotaxis, but not cell cycle progression.

Keywords
  • NAD(P)H oxidase
  • Src
  • Atherosclerosis
  • Platelet-derived growth factor
  • ROS

1. Introduction

Reactive oxygen species (ROS) play an important role in the pathogenesis of cardiovascular diseases including hypertension, atherosclerosis, myocardial hypertrophy, and restenosis [7]. ROS are often considered as toxic products acting mainly as pathogens. However, they appear to contribute to various cellular functions as second messengers and are involved in the regulation of vascular tone, migration, and apoptosis. NAD(P)H oxidases are the predominant source of ROS in the vascular wall [22]. Expression and activity of these enzymes are regulated via growth factors like angiotensin II, transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF) [22].

The mechanisms of superoxide production via NAD(P)H oxidases have been extensively studied in phagocytes. In these cells, the enzyme represents a multiprotein system consisting of two membrane-bound subunits, gp91phox (also termed Nox2) and p22phox, which together form the flavocytochrome b558. The enzyme's activity is regulated by the cytosolic components p47phox, p67phox, p40phox, and the Rho GTPase Rac2 in human neutrophils. Upon activation, p47phox becomes phosphorylated and mediates translocation of the p47phox/p67phox/p40phox complex to the membrane [42]. Rac2 separates from a GDP dissociation inhibitor and co-migrates to the membrane after exchange of GDP for GTP. The assembled enzyme complex is now able to catalyze the one-electron reduction of molecular oxygen to form superoxide.

In non-phagocytes, NAD(P)H oxidases differ from the phagocytic enzyme. Differences include activation kinetics, constitutive activity, and the amount of superoxide, which is produced mainly intracellularly [22]. A structural difference is the occurrence of homologues of gp91phox (Nox1-5), the large membrane-bound subunit of cytochrome b558 [15,28,35,40]. The significance of these novel homologues is not yet fully understood, as is the expression pattern in various cell types. In vascular smooth muscle cells (VSMC), expression of Nox1, Nox4, and Nox5 has been detected [9,17,20,35]. Overexpression of Nox1 and Nox4 in VSMC suggests that Nox1 has mitogenic activity, whereas Nox4 is involved in cellular senescence [23]. Nox1 is upregulated by growth factors, whereas Nox4 is downregulated by these agonists [23]. Consequently, these data suggest an opposing, or at least distinct function of individual Nox isoforms in redox signaling.

Because NAD(P)H oxidases are involved in the development and progression of a wide spectrum of diseases, including cancer [37], Alzheimer's [34] and cardiovascular diseases, they may represent a significant therapeutic target. Several inhibitors of NAD(P)H oxidases have been identified, although most of them encounter difficulties in practice. Peptidic inhibitors like gp91ds-tat are useful in experimental procedures [19,30], but cannot be administered orally. Furthermore, gp91ds-Tat is unlikely to inhibit NAD(P)H oxidases that use Nox proteins apart from Nox2. Aminoethyl-benzenesulfono-fluoride (AEBSF) prevents the binding of flavocytochrome b558 to p47phox [10], but is a serine protease inhibitor with many additional effects. Diphenylene iodonium (DPI) is an unspecific inhibitor of all flavoenzymes, which–apart from NAD(P)H oxidases–include xanthine oxidase and cytochrome P450 enzymes [27]. Apocynin, extracted from Picrorhiza kurroa, prevents the formation of the active oxidase complex [36] and has been studied in a number of animal models of asthma, inflammation, and atherosclerosis [6]. Although it is effective when administered orally [2], it has to be administered in extremely high concentrations. Consequently, apocynin is not specific for NAD(P)H oxidases, but rather influences other events such as Thromboxane A2 formation [11] and the induction of AP-1 transcription factor [21].

In the present study, we characterized the novel Nox inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870) in VSMCs. We investigated its effect on PDGF-BB-dependent NAD(P)H oxidase activation, intracellular ROS production, and growth factor-dependent chemotaxis and proliferation, which are critically involved in pathogenic processes like tumorigenesis and atherogenesis. Furthermore, we related the effects to downstream signaling events such as activation of Src kinase, Akt, and Erk 1/2.

2 Materials and methods

2.1 Cell culture

Vascular smooth muscle cells (VSMC) were isolated from rat thoracic aorta (Wistar Kyoto; 6–10 weeks old; Charles River Wega GmbH, Sulzfeld, Germany) by enzymatic dispersion as described [8]. Cells were grown in a 5% CO2 atmosphere at 37 °C in DMEM supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 1% nonessential amino acids (100 ×), and 10% FCS. Experiments were performed with cells from passages 5–10 (proliferation and migration assays) and 5–15 (biochemical analyses).

2.2 Determination of intracellular reactive oxygen species

ROS production was investigated by 2′,7′-dichlorofluorescein (DCF) fluorescence using confocal laser scanning microscopy. Subconfluent cells plated on glass carriers were starved by serum deprivation for 24 h and stimulated as indicated. Cells were then washed once in buffer E1 (containing NaCl 135 mmol/l, KCl 5.4 mmol/l, CaCl2, 1.8 mmol/l MgCl2 1 mmol/l, glucose 10 mmol/l, HEPES 10 μmol/l, pH 7.5, 37 °C) and incubated in the dark for 30 min in the same buffer containing 10 μmol/l 2′,7′-dichloro-dihydro-fluorescein-diacetate (H2DCF-DA, Molecular Probes, Eugene, OR, USA). H2DCF-DA is a non-polar compound that readily diffuses into cells, where it is hydrolyzed to the non-fluorescent polar derivate H2DCF and thereby trapped within the cell. In the presence of a proper oxidant, H2DCF is oxidized to the highly fluorescent DCF [13]. Culture dishes were transferred to a Zeiss Axiovert 135 inverted microscope (Carl Zeiss, Jena, Germany), equipped with a 25 ×, numerical aperture 0.8, oil-immersion objective (Plan-Neofluar, Carl Zeiss) and Zeiss LSM 410 confocal attachment, and ROS generation was detected as a result of H2DCF oxidation (excitation, 488 nm; emission longpass LP515-nm filter set). 512 × 512 pixel images were collected by single rapid scans. In three separate experiments, groups of 25 cells each were randomly selected from the image, and fluorescent intensity was measured. The relative fluorescence intensity is expressed as mean±SEM from three independent experiments.

2.3 NAD(P)H oxidase activity

NAD(P)H oxidase activity was measured by lucigenin-enhanced chemiluminescence in a 50 mmol/l phosphate buffer (buffer A), pH 7.0, containing 1 mmol/l EGTA, protease inhibitors (Complete, Boehringer Mannheim, Germany), 150 mmol/l sucrose, 5 μmol/l lucigenin (Sigma, Deisenhofen, Germany), and 250 μmol/l NADPH as substrate. Quiescent cells were starved by serum deprivation for 24 h and treated as indicated, washed twice with ice-cold phosphate buffered saline (PBS), pH 7.4, and harvested. After low spin centrifugation, the pellet was resuspended in ice-cold buffer A, lacking lucigenin and substrate. Then, the cells were lysed using a Dr. Hielscher 200s sonificator. The total protein concentration was determined using a Bradford assay (BioRad, Heidelberg, Germany) and adjusted to 1 mg/ml. 100 μl aliquots of the protein sample were measured over 6 min in quadruplicates using NADPH (100 μM) as substrate in a scintillation counter (Berthold Lumat LB 9501). Data were collected at 2 min intervals in order to measure relative changes in NAD(P)H oxidase activity.

2.4 Chemotaxis assay

PDGF-dependent chemotaxis was assayed utilizing a 48-well modified Boyden chemotaxis chamber (NeuroProbe Inc., Baltimore, MD) and PVP-free polycarbonate filters (8 μm pore size) (Poretics Corp., Livermore, CA) as described previously [31]. Briefly, the lower wells of the chamber were filled with DMEM supplemented with 10 ng/ml PDGF-BB or vehicle in the presence or absence of VAS2870. The filters were coated with 50 mg/ml rat type I collagen (Collaborative Biomedical Products, Bedford, MA) and fixed atop the bottom wells. VSMCs were trypsinized, washed and diluted in DMEM to a final concentration of 4 × 105 cells per ml. 50 μl of this cell suspension were placed into the top wells. In each experiment, at least 6 of the chamber's 48 wells were used for each condition examined. The chamber was incubated for 4 h at 37 °C in a 5% CO2 atmosphere.

Following incubation, the chamber was disassembled, the cells on the upper surface of the filter were removed, and the cells on the lower surface fixed and stained with Diff-Quick (Baxter Healthcare Corp., Miami, FL). Chemotaxis was quantified by counting the number of cells on the lower surface of the filter in each well using a grid containing 100 non-overlapping fields. The total number of cells per 100 fields was 5–20 in resting cells, and 80–150 in responding cells. The response being measured was primarily chemotaxis, since including PDGF in the top and bottom chamber reduced the number of migrated cells by approximately 70% [31].

2.5 DNA synthesis assay

DNA synthesis was measured by a 5-bromo-deoxyuridine (BrdU)-incorporation assay as described [32]. Briefly, cells were cultured in 96-well-plates to ∼90% confluence, washed, fed with DMEM and starved for 24 h. PDGF-BB was added to cells for 18 h at the indicated concentrations in the absence or presence of various concentrations of VAS2870. The BrdU-incorporation assay was carried out according to the manufacturer's specifications (Roche) with an incorporation time of 5 h.

2.6 Immunoprecipitation and Western blot analysis

Quiescent VSMCs were left resting or stimulated with 20 ng/ml PDGF-BB for 10 min in the presence or absence of VAS2870 as indicated. The βPDGFR was immunoprecipitated as previously described [32]. βPDGFR immunoprecipitates were resolved on a 7.5% SDS-polyacrylamide electrophoresis gel (PAGE), and subjected to Western blot analysis using antisera that recognize phosphotyrosine, as described [31].

2.7 Activation of downstream signaling molecules

To examine the influence of VAS2870 on the ligand-induced activation of downstream signaling molecules, quiescent VSMC were stimulated with PDGF-BB (20 ng/ml) for 10 min, lysed and similar amounts of total cell lysates were subjected to Western blot analysis using antisera against phospho-Erk 1/2 (Thr202/Tyr204), phospho-Akt (Ser 473) and phospho-Src (Tyr 416).

2.8 Cell viability assay

Cells were cultured in 96-well plates, starved by serum deprivation for 24 h and treated with various concentrations of VAS2870 as indicated. Cell viability was assessed using a membrane integrity assay according to manufacturer's instructions (CytoTox-One, Promega).

2.9 VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine)

VAS2870 was provided by vasopharm BIOTECH GmbH, Würzburg, Germany. It represents a drug-like small molecule with a molecular weight of 360,4. VAS2870 was characterized by NMR and mass spectrometry (1H nmr (DMSO-D6): δ 5.85 (s, 2H, CH2), 7.3–7.4 (m, 5H, Ph), 7.5–7.6 (m, 2H, Ar), 7.85 (d, 1H, Ar), 7.95 (d, 1H, Ar), 8.95 (s, 1H, H-5). ms: (+APCl) m/z 361 [M+H]+). VAS2870 has been identified by NAD(P)H oxidase specific high-throughput screening [39] (Fig. 1A). The effect of VAS2870 on the superoxide formation was investigated in a cell-free system with membranes and cytosol from human neutrophils as described in [26]. An IC50-value of 10.6 μM was detected (Fig. 1B). ROS production in HL-60 cells was measured as described in [39]. IC50-values in these cells were slightly lower (2 μM) than those in the cell-free system. While the exact acting site of the compound remains elusive, VAS2870 did not affect PMA-stimulated translocation of p47phox in human PMN (R. Brandes, unpublished observation, 2004).

Fig. 1

Characteristics of the novel Nox inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870). (A) Chemical structure. (B) Concentration-dependent effect of VAS2870 on NAD(P)H oxidase activity in a cell-free system with membranes and cytosol from human neutrophils (IC50 10.6 μM).

2.10 Materials and antibodies

PDGF-BB was purchased from Promo Cell. The antibody against RasGAP (69.3) was a kind gift from Andrius Kazlauskas (Harvard Medical School, Boston). The phospho-specific p42/44 Erk antibody was from New England Biolabs, the phospho-specific Src and Akt antibodies were from Cell Signaling.

2.11 Statistical analysis

All data are expressed as means±SEM. Statistical analysis was evaluated by non-parametric analysis. p<0.05 was considered significant.

2.12 Ethical statement

This study conforms to NIH Guidelines.

3 Results

3.1 VAS2870 potently inhibits PDGF-mediated NAD(P)H oxidase activation and intracellular ROS formation in VSMC

To investigate the influence of VAS2870 on PDGF-BB-dependent activation of NAD(P)H oxidase in VSMCs, lucigenin-enhanced chemiluminescence was measured in cell lysates using NADPH as substrate. Stimulation with PDGF-BB (50 ng/ml) led to a time-dependent increase of NAD(P)H oxidase activity to 219±27% at 1 h and 229±9% at 2 h (Fig. 2A). As demonstrated in Fig. 2B, pretreatment of the cells with VAS2870 (20 min before PDGF stimulation) completely abolished PDGF-BB-dependent NAD(P)H oxidase activation.

Fig. 2

VAS2870 suppresses PDGF-BB-induced activation of NAD(P)H oxidase. NAD(P)H oxidase activity was assessed as lucigenin-enhanced chemiluminescence (5 μM) in homogenates of VSMC using NADPH as substrate. Values represent the percentual increase of chemiluminescence over buffer stimulation, and are expressed as means±SEM of three independent experiments, each performed in quadruplicate. (A) PDGF-BB (50 ng/ml) time-dependently increased NAD(P)H oxidase activity (*p<0.05, §p<0.01 vs. buffer-stimulated cells). (B) Pretreatment of cells with VAS2870 (20 min prior to PDGF-BB) completely abolished PDGF-dependent NAD(P)H oxidase activation at concentrations of 10 and 20 μM (**p<0.05 vs. PDGF-BB-stimulated cells).

VSMC are known to produce mainly intracellular ROS in response to activation of the β-PDGF receptor (βPDGFR). In order to determine the influence of VAS2870 on PDGF-induced ROS liberation, intracellular ROS levels were measured by confocal laser scanning microscopy using the fluorescent dye H2DCF-DA. Stimulation of the βPDGFR with a saturating concentration of PDGF-BB (50 ng/ml) led to a dramatic increase of intracellular ROS levels to 362±54% within 2 h (Fig. 3A and B). To prove that VAS2870 does not act as a scavenger of ROS, we also performed a xanthine/xanthine-oxidase assay. VAS2870 at concentrations up to 50 μM did not influence xanthine-oxidase-mediated superoxide production as determined by cytochrome C reduction (not shown). Consequently, VAS2870 does not act as an antioxidant. The inhibition of NAD(P)H oxidase activation by VAS2870 resulted in a complete abrogation of the PDGF-dependent intracellular ROS production (Fig. 3A and B). Thus, VAS2870 is able to sufficiently inhibit PDGF-dependent NAD(P)H oxidase activation and subsequent production of ROS in VSMC.

Fig. 3

VAS2870 prevents PDGF-mediated intracellular production of ROS. ROS production was assessed by confocal laser scanning microscopy using 2,7-dichloro-dihydro-fluoresceine-diacetate (10 μM), which specifically stains intracellular ROS. (A) Shown are representative original rapid confocal laser scans. PDGF-BB (50 ng/ml) led to increased intracellular ROS levels within 2 h, and this effect was completely abolished by preincubation with VAS2870 (10 and 20 μM). (B) Quantitative analyses of three independent experiments as in A, each performed as triplicate. Data are expressed as percentual increase of DCF fluorescence over buffer stimulation and represent means±SEM. Stimulation of the âPDGFR with PDGF-BB (50 ng/ml) led to an increase of relative DCF fluorescence to 362±54% at 2 h (*p<0.01 vs. buffer-stimulated cells). Pretreatment with VAS2870 completely abolished the PDGF-BB-mediated production of ROS (**p<0.05 vs. PDGF-BB-stimulated cells).

3.2 VAS2870 inhibits PDGF-BB-mediated migration of VSMC but does not affect cell cycle progression

PDGF-dependent migration and proliferation of cells are critical pathogenic events in proliferative disease progression. Because ROS are thought to be involved in growth-factor-mediated signaling events in VSMC, we next explored the influence of VAS2870 on PDGF-dependent cellular responses.

Chemotaxis of VSMC was measured in the modified Boyden chamber. PDGF-BB (10 ng/ml) led to a robust increase of cell-migration to 417±22% compared to unstimulated cells. Fig. 4A demonstrates that pretreatment with VAS2870 dose-dependently inhibited this effect and completely abolished PDGF-dependent migration at a concentration of 10 μM.

Fig. 4

VAS2870 suppresses PDGF-mediated chemotaxis, but not cell cycle progression in VSMC. (A) PDGF-BB-mediated cell migration was assessed in the modified Boyden chamber. Values represent the percentual increase of migrated cells over buffer stimulation (100%). Data are expressed as means±SEM of three independent experiments. Stimulation with PDGF-BB (10 ng/ml) led to an increase in cell migration to 417±22% (*p<0.001 vs. buffer). Preincubation with VAS2870 dose-dependently suppressed this effect and completely abolished PDGF-induced chemotaxis at 10 μM (**p<0.05 vs. PDGF-BB). (B) PDGF-dependent DNA synthesis was detected by BrdU incorporation. Values represent the percentual increase of BrdU incorporation over buffer stimulation (100%). Data are expressed as means±SEM of a series of seven independent experiments, each performed as quintuplicate. PDGF-BB led to an increase in DNA synthesis to 321±42% (20 ng/ml, *p<0.01 vs. unstimulated cells). VAS2870 in various concentrations (0.1–10μM) had no influence on PDGF-dependent DNA synthesis (*p<0.05 vs. buffer).

PDGF-BB is known to act as a potent mitogen on various cell types including VSMC, and ROS are thought to be involved in the signaling processes leading to cell proliferation. To investigate, whether inhibition of NAD(P)H oxidase/ROS liberation by VAS2870 would affect PDGF-dependent cell cycle progression in VSMC, we measured the incorporation of 5-bromo-deoxyuridine (BrdU) upon PDGF stimulation in the absence or presence of VAS2870. Fig. 4B demonstrates that PDGF-BB (20 ng/ml) led to a concentration-dependent increase in DNA synthesis to 321±42% compared to unstimulated cells. Surprisingly, the preincubation of the cells with various concentrations (0.1–10 μM) of VAS2870 had no influence on PDGF-dependent DNA synthesis.

In summary, these data demonstrate that inhibition of NAD(P)H oxidase activity and intracellular ROS liberation by VAS2870 abolished PDGF-dependent migration of VSMC, whereas it had no influence on cell cycle progression initiated by the βPDGFR. Hence, our finding indicates that some PDGF-induced cellular responses require redox-sensitive signaling pathways, whereas others do not.

3.3 Preincubation with VAS2870 has no influence on PDGF-dependent tyrosine phosphorylation of the βPDGFR or cell viability

In order to explore the possibility that the inhibitory effects of VAS2870 on PDGF-dependent chemotaxis of VSMC were due to direct inhibition of receptor activation, as described for other compounds (e.g. the Src inhibitor PP1 [43]), we monitored the influence of VAS2870 on the ligand-induced tyrosine phosphorylation of the βPDGFR. Quiescent VSMC were stimulated with PDGF-BB (20 ng/ml), the βPDGFR was immunoprecipitated, and Western blot analyses were performed using antibodies that recognize phospho-tyrosine. As shown in Fig. 5, stimulation with PDGF robustly increased the phosphotyrosine content of the receptor. However, while similar amounts of receptor were present in all of the samples (top band), VAS2870 at various concentrations had no influence on the phosphotyrosine content (bottom band), indicating that it has no effect on the ligand-induced receptor activation. Consequently, inhibitory effects of VAS2870 on cellular functions cannot be explained by a direct interaction with the βPDGFR.

Fig. 5

The ligand-induced tyrosine phosphorylation of the βPDGFR is not influenced by VAS2870. To monitor a possible influence of VAS2870 on the ligand-induced activation of the βPDGFR, the βPDGFR was immunoprecipitated from PDGF-stimulated cells that had been preincubated with VAS2870. Immunoprecipitates were subjected to Western blot analyses using antisera that recognize phospho-tyrosine (P-Y; bottom panel in A) or the βPDGFR (top panel in A). Densitometric analyses of three independent experiments are demonstrated in B. Values are expressed as means±SEM. They are normalized for the βPDGFR content of immunoprecipitates and represent the percentual changes of tyrosine phosphorylation upon stimulation with PDGF-BB (50 ng/ml, 5 min).

To ensure that VAS2870 did not exert cytotoxic effects on VSMC, we determined cell viability using a lactate dehydrogenase (LDH)-assay as a parameter for membrane integrity. At the used concentrations (0.1–20 μM), VAS2870 had no influence on cell viability. However when higher concentrations were administered, cytotoxicity increased above 30% compared to chemically lysed cells (100%) (not shown). In our experiments, the highest concentration of VAS2870 used was 20 μM.

3.4 PDGF-dependent activation of Src–but not Erk 1/2 and Akt–is suppressed by VAS2870

The fact, that inhibition of intracellular ROS production by VAS2870 abolishes PDGF-dependent chemotaxis, but not DNA synthesis, suggests that at least some of the signaling pathways that are required for the chemotactic response are redox-sensitive. Using a series of PDGFR mutants, we have previously shown that PDGFRs elicit their mitogenic signal via activation of Phospholipase C γ (PLCγ) and Phosphatidyl-inositol-3-kinase (PI3K), but not Src family kinases, whereas the chemotactic response also requires PI3K and PLCγ, but is particularly dependent on Src activation ([31], Vantler and Rosenkranz, unpublished observations). These previous findings identified Src as a potential candidate that may explain the differential effects of VAS2870 on PDGF-dependent migration versus proliferation. Therefore, we investigated whether Nox inhibition attenuates PDGF-induced Src activation and compared the response to other relevant downstream signaling events such as Akt and Erk 1/2. To this end, quiescent VSMC were stimulated with PDGF-BB in the presence or absence of VAS2870, the cells were lysed, and total cell lysates were subjected to Western blot analyses using phospho-specific antibodies directed against Src, Akt, and Erk 1/2.

As shown in Fig. 6A, PDGF-BB (20 ng/ml) led to rapid phosphorylation of Akt and Erk 1/2. Pretreatment with VAS2870 had no influence on these events, indicating that these signaling pathways do not require NAD(P)H oxidase activity or intracellular ROS. Stimulation of the cells with PDGF-BB also led to phosphorylation of Src at the tyrosine residues required for Src activation (Fig. 6B). However, when PDGF-BB was added in the presence of VAS2870, the Nox inhibitor concentration-dependently reduced PDGF-dependent Src activation and completely abolished it at a concentration of 10 μM. Consequently, it is likely that the inhibitory effect of VAS2870 on PDGF-BB-dependent chemotaxis is due to the lack of efficient Src activation. Furthermore, our data indicate that PDGF-BB-mediated activation of Akt and Erk 1/2 is independent of NAD(P)H oxidase and ROS. To rule out the possibility that VAS2870 directly affected PDGF signaling to Src rather than through inhibition of ROS production, we performed additional experiments using the direct ROS scavenger polyethylene glycol (PEG)-catalase instead of VAS2870. Fig. 7 demonstrates that PEG-catalase–like VAS2870–inhibited PDGF-dependent phosphorylation of Src, but not Erk 1/2 and Akt, indicating that the Src signaling pathway is indeed redox-sensitive. Similar results were also obtained when the cells were stimulated with angiotensin II instead of PDGF (not shown).

Fig. 7

PEG-catalase blocks PDGF-dependent phosphorylation of Src, but not of Erk 1/2 and Akt. To assess the influence of ROS on the activation of downstream signaling molecules, VSMC were pretreated with various concentrations of PEG-catalase as indicated, and stimulated with PDGF-BB (20 ng/ml). The cells were harvested, and equal amounts of total cell lysates were subjected to Western blot analyses using phospho-specific antibodies that recognize activating phosphorylation of Akt, Erk 1/2 (A), or Src (B). Presented are representative Western blots (top bands (RasGAP) represent lysate controls) and densitometric analyses of at least three independent experiments (n=3 for p-Akt and p-Erk 1/2, n=4 for p-Src). Values are expressed as means±SEM. They are normalized for the protein content of lysates and represent the percentual changes of protein phosphorylation upon stimulation with PDGF-BB (*p<0.05 vs. PDGF-BB).

Fig. 6

PDGF-dependent phosphorylation of Src, but not of Erk 1/2 and Akt, is attenuated by VAS2870. To assess the influence of Nox inhibition on PDGF-dependent phosphorylation of downstream signaling molecules, VSMC were pretreated with various concentrations of VAS2870 as indicated, and subsequently stimulated with PDGF-BB (20 ng/ml) for 5 min. The cells were harvested, and equal amounts of total cell lysates were subjected to Western blot analyses using phospho-specific antibodies, that recognize activating phosphorylation of Akt, Erk 1/2 (A), or Src (B). Demonstrated are representative Western blots (top bands (RasGAP) represent lysate controls) and densitometric analyses of at least three independent experiments (n=6 for p-Akt and p-Erk 1/2, n=3 for p-Src). Values are expressed as means±SEM. They are normalized for the protein content of lysates and represent the percentual changes of protein phosphorylation upon stimulation with PDGF-BB (*p<0.05 vs. PDGF-BB).

In summary, our data demonstrate that NAD(P)H oxidase and NAD(P)H oxidase-derived ROS are critical for PDGF-dependent migration of VSMC, but are not involved in the signaling mechanisms that lead to proliferation of these cells. Since Src is absolutely required for PDGF-dependent chemotaxis, but not DNA synthesis, the distinct influence of Nox inhibition by VAS2870 on these cellular events may be explained by VAS2870-dependent inhibition of Src activation.

4 Discussion

In the present study, we have determined the influence of the novel Nox inhibitor VAS2870 on NAD(P)H oxidase activity, ROS production and functional effects mediated by growth factors in VSMC. We found that VAS2870 is effective to suppress PDGF-BB-dependent activation of NAD(P)H oxidase and subsequent production of intracellular ROS. Furthermore, VAS2870 suppressed PDGF-BB-dependent chemotaxis, but not DNA synthesis. These findings can neither be explained by direct inhibition of PDGFR tyrosine phosphorylation (activation), nor by antioxidant activity or cytotoxic effects of the Nox inhibitor. Instead, VAS2870 abolished specific signaling molecules such as Src, which appear to be redox-sensitive.

The role of ROS as a signaling molecule in cell migration has been reported by several groups [18,44]. Furthermore, it has been shown that ROS are involved in the activation of the non-receptor tyrosine kinase Src. Src subsequently leads to activation of phosphoinositide-dependent kinase 1 (PDK1) and p21-activated kinase 1 (PAK1). Here, we demonstrate that the Nox inhibitor VAS2870 concentration-dependently abolished βPDGFR-dependent migration of VSMC. Complete inhibition was achieved by a concentration of 10 μM. These findings are consistent with other reports demonstrating that antioxidants like N-acetylcysteine (NAC) and ebselen inhibit PDGF-dependent chemotaxis in VSMC [45]. In addition, our observation that VAS2870 suppressed PDGF-dependent Src activation is consistent with recent publications demonstrating an involvement of ROS in the activation of Src [24,45].

In contrast to chemotaxis, pretreatment with VAS2870 did not influence PDGF-BB-mediated cell cycle progression, as determined in a BrdU incorporation assay. The role of ROS in growth factor-mediated cell proliferation is not yet fully understood. There is a substantial number of publications indicating that antioxidants like NAC, tiron or ebselen, the flavoprotein inhibitor DPI or overexpression of catalase reduce agonist-dependent proliferation in various cell types [25,29,38,41]. It has to be noted that the concentrations used e.g. for NAC vary between 100 μM [45] and 20 mM [25], representing a 200-fold range. Furthermore, a basal level of ROS may be necessary to keep proteins in their normal redox state. Reducing the majority of intracellular signal transducing molecules might lead to changes in many signaling cascades, and does not represent a physiologic state. Additionally, it has been shown that overexpression of catalase promotes apoptosis [5]. Other groups have used more specific methods, e.g. expression of dominant-negative p22phox or application of antisense oligonucleotides against Nox1 or Nox4. Nevertheless, the results from these studies are not consistent. Treatment with antisense p22phox led to reduced thymidine incorporation in coronary vascular endothelial cells [1], reduced proliferation of human airway smooth muscle cells [3] and melanoma cells [4]. Conversely, Geist et al. have reported that antisense approaches against Nox1 in colon cancer samples (cells which overexpress Nox1) did not affect proliferation [16]. Our data indicate that NAD(P)H oxidase-derived ROS are not involved in PDGF-induced proliferation of VSMC. Consistent with this finding, PDGF-mediated proliferation was not affected in p47phox-deficient fibroblasts compared to wildtype cells (Bäumer and Rosenkranz, unpublished observations).

In order to further examine our finding that NAD(P)H oxidase-derived ROS are involved in PDGF-BB-mediated chemotaxis, but not proliferation of VSMC, we performed Western blot analyses of potentially redox-sensitive signaling molecules like Akt, Erk 1/2, and especially Src. In our model, PDGF-BB led to the activation of Akt and Erk 1/2, as expected. Pretreatment with various concentrations of VAS2870 as well as PEG-catalase had no influence on this observation. In the literature, the influence of ROS on Erk activation is controversial. Several groups have shown that pretreatment of cells with antioxidants prevents Erk activation [25]. Others have repeatedly shown the opposite, even in VSMC [45]. For Akt, a downstream effector of PI3K, an influence of ROS has been reported. The stimulation of rat fibroblasts with hydrogen peroxide led to the activation of Akt [12]. Furthermore, it has been shown that PDGF-BB and Angiotensin II can activate Akt in a ROS-dependent manner [45]. The latter has been shown using antioxidants. In our system, the phosphorylation of Akt at Ser473 was independent of ROS and NAD(P)H oxidase activity.

In our studies, PDGF-BB led to phosphorylation of Src, and pretreatment of VSMC with VAS2870 or PEG-catalase concentration-dependently suppressed this effect. For Src family kinases, a feed-forward mechanism has been proposed. Small amounts of ROS are able to activate Src, which itself is a known activator of NAD(P)H oxidases. The activation of the enzyme leads to the production of ROS, which in turn promotes further activation of Src [33]. Recently, our group has identified the critical signaling molecules for βPDGFR-dependent proliferation and chemotaxis. We could demonstrate that PI3K and PLCγ are the only two effectors critical for proliferation of VSMC. Furthermore, these molecules contribute to PDGF-BB-mediated migration. However, the main effector of βPDGFR-induced migration is Src, as demonstrated by chimeric CSF1R/βPDGFR mutants that lack specific binding sites for receptor-associated signaling molecules [31] (Vantler and Rosenkranz, unpublished observations). When taken together, the absolute requirement of Src for PDGF-mediated chemotaxis and the present finding that Src phosphorylation is inhibited by VAS2870 are consistent with the idea that suppression of ROS-dependent Src activation affects chemotaxis, but not DNA synthesis. Furthermore, our data are in line with reports from other investigations.

Lin et al. found that resveratrol, an antioxidant present in red wine, inhibited VEGF-dependent migration but not proliferation at concentrations between 1 and 2.5 μM in human umbilical vein endothelial cells (HUVECs). The authors found that resveratrol only affected the activation of Src, but not of Akt and Erk1/2. Furthermore, preincubation with NAC prevented the activation of Src and reduced VEGF-mediated migration of HUVECs [24]. In summary, the role of ROS and Src in growth factor-induced chemotaxis has been repeatedly demonstrated.

Conversely, there is inconsistency in literature concerning the role of Src in cell proliferation. While some studies indicate that Src is required for cell cycle progression [14], our present findings using a Nox inhibitor indicate that PDGF-mediated proliferation of VSMC occurs independently of Src. Furthermore, Lin et al. have shown that Src-inhibition by resveratrol was not associated with an antiproliferative effect [24].

In summary, we conclude that NAD(P)H oxidase-derived ROS are involved in PDGF-mediated migration, but not proliferation of VSMC. The ligand-induced activation of Src, a key player in chemotactic signal transduction by the PDGFR, is suppressed by the Nox inhibitor VAS2870, whereas other signaling pathways that lead to activation of Akt and Erk 1/2, are not affected. The suppression of specific signaling pathways by sufficient Nox inhibition is likely to account for the differential effects on distinct cellular responses.

Footnotes

  • Time for primary review 23 days

  • This paper was handled by special Guest Editor Andrew C. Newby, Bristol, UK.

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View Abstract